A novel synthesis method for large-pore, well-aligned 2D hexagonal mesoporous silica thin films is reported. The alignment was achieved by confinement in poly(dimethylsiloxane) (PDMS) microgrooves without the necessity of additional forces (such as electric fields). We describe the influence of various experimental conditions including the way the grooves are filled, surface modification at the solid/liquid interfaces, and the height-to-width ratio of the microgrooves on mesopore alignment. With this technique, highly oriented mesoporous silica channels can be obtained at a length scale of several millimeters. For a nondestructive, detailed, and wide-ranging structural and dynamic characterization of the as-synthesized mesochannel silica network, dye molecules were incorporated into the channels at concentrations suitable for single-molecule microscopy. A “maximum projection” of individual frames recorded with a fluorescence microscope immediately gives a global overview (“map”) of the pore structure, thus providing direct feedback for tuning synthesis conditions. In addition, deeper insights into the real nanoscale structure of the mesoporous silica framework were obtained through high-accuracy single-molecule tracking experiments. The high spatial accuracy of the experiments allowed for the direct observation of jumps of single dye molecules between individual channels in the mesoporous silica host. Nevertheless, due to the low concentration of defects, the diffusion could be described as a 1D random walk where the molecules diffuse along the highly oriented, parallel channels and sometimes switch from channel to channel through small defects in the pore walls. Furthermore, it could be shown with single-molecule microscopy that template removal and calcination of the aligned films results in an increased defect concentration; however, the overall order of the structures remained intact.